Melphalan derivatives and their use as cancer chemotherapeutic drugs

ABSTRACT

The invention refers to new alkylating di- and tripeptides based on a melphalan unit, and one or two additional amino acids or amino acid derivatives, which can be used in the treatment of carcinogenic diseases. Further, the invention refers to a pharmaceutical composition comprising the alkylating peptides of the invention.

The present invention relates to new melphalan derivatives useful asalkylating cancer chemotherapeutic drugs.

BACKGROUND OF THE INVENTION

Cancer is a major and often fatal disease. Accordingly, efforts todevelop new therapies for cancer is a constantly ongoing effort of theresearch society. The vast majorities of cancers are present as solidtumours, e.g. lung cancer, breast cancer, prostate cancer, while therest are hematological and lymphoid malignancies, e.g. leukemias andlymphomas.

Chemotherapy is used in attempts to cure or palliate the disease. Inmost cases this therapy is delivered in the form of combinationchemotherapy, when two or more drugs having different modes of actionare used together in order to optimise the antitumoural effect and tominimise side effects. The results obtained with chemotherapy varyaccording to tumour type. Some tumours are very sensitive and thetreatment has then a high probability of leading to cure. Examples ofthis type of tumours are acute leukemias, malignant lymphomas,testicular cancer, chorion carcinomas and Wilms tumour. In another groupof tumours chemotherapy can result in good palliation and prolongedsurvival. Examples of such tumours are breast cancer, colo-rectalcancer, ovarian cancer, small-cell lung cancer, bladder cancer, multiplemyeloma, and chronic leukemias of both lymphatic and myeloid type.Primary drug resistant tumours are for instance malignant glioma,melanoma, prostate cancer, sarcomas and gastrointestinal tumours otherthan colo-rectal cancers.

Alkylating agents, such as drugs derived from nitrogen mustard, that isbis(2-chloroethyl)amine derivatives, are used as chemotherapeutic drugsin the treatment of a wide variety of neoplastic diseases. These drugsall act by covalent interaction with nucleophilic heteroatoms in DNA orproteins. It is believed that these difunctional agents are able tocrosslink a DNA chain within a double helix in an intrastrand orinterstrand fashion, or to crosslink between DNA and proteins. Thecrosslinking results in inhibitory effects on DNA replication andtranscription with subsequent cell death. The drugs can be used assingle agents or in combination with other antineoplastic agents.Alkylating agents seem to have some propensity for fast growing tissues.They exert effects in a broad spectrum of tumours. Side effects aremainly restricted to bone marrow and at very high doses also to thegastrointestinal tract.

Melphalan, or p-bis-(2-chloroethyl)-aminophenylalanine, is a conjugateof nitrogen mustard and the amino acid phenylalanine, which wassynthesised in the mid 1950s (U.S. Pat. No. 3,032,584). This classicalkylating substance soon became a valuable drug in the chemotherapeuticfield and is still of importance in the treatment of for examplemyeloma. Melphalan was originally developed as a selective cytotoxicagent against melanoma cells, which utilise large amounts ofphenylalanine in melanin synthesis. Clinical use of melphalan in thetreatment of metastatic melanomas has, however, had limited efficacy.

In the search for a more selective action on malignant cells melphalananalogues have been synthesised. Sarcolysine,m-bis-(2-chloroethyl)aminophenylalanine, was obtained by shifting thebis-(2-chloroethyl)-amino group from the para- to the meta-position ofphenylalanine. By covalent conjugation of different amino acids at theamino and carboxylate groups of sarcolysine a peptide mixture known asPeptichemio® (PTC) was prepared. PTC consisted of six different peptides(de Barbieri, “Proceedings of the symposium on Peptichemio”, Milan, Nov.18, 1972). PTC was subsequently shown to be active on several tumourtypes as well as on tumours resistant to treatment with alkylatingagents including melphalan and entered clinical trials with promisingresults. For the understanding of the effects and usage of PTC a seriousdisadvantage was the fact that it is a mixture of six peptides. Thecytotoxic effects of each of the different peptides contained in PTCwere therefore measured separately (Lewensohn et al., AnticancerResearch 11: 321–324 (1991)) and a wide variation in the cytotoxicitiesof the peptides was found. One of the peptides,L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine ethyl esterhydrochloride (P2) turned out to be more toxic to RPMI 8322 melanomacells than any of the other peptides.

PRIOR ART

Lopatin et al. (CA 79:100709, Farmakol. Toksikol. (Moscow), 1973, 36(4),479–480) discloses that administration of the melphalan derivativesasaley and astyron inhibited sarcoma 45 growth in rats. Astyron, orN-acetyl-L-melphalanyl-L-tyrosine ethyl ester, as well as asaley, orN-acetyl-L-melphalanyl-L-valine ethyl ester, have an acetylated aminogroup, which generally means that the cytotoxicity of the compound totumour cells has been considerably reduced compared to the nonacetylated form.

Romanova et al. (CA 66:27517, Vopr. Med. Khim, 1966, 12 (6), 586–91)refers to a compound called sarcolysine or salin, which according toWestern nomenclature should rather be named L-melphalanyl-L-valine. Thiscompound disrupted oxidative phosphorylation in mitochondria from ratliver homogenates and Jensen sarcoma cells.

There is a slight confusion as to the nomenclature of the melphalanderivatives. When, in 1955, a British research group reported thesynthesis and cytotoxic activity of a phenylalanine derivativesubstituted with a bis-(2-chloroethyl)amino group in the para-positionof the aromatic ring, the DL, L and D isomers were named merphalan,melphalan and medphalan, respectively. A Russian group at the same timeindependently named the racemic form (DL) of4-[bis-(2-chloroethylamino)]-phenylalanine sarcolysine. Later the termsarcolysine started to be used also for the meta-phenylalanine mustard.This name confusion has continued, but today melphalan and sarcolysineare normally used for the para- and meta-derivatives, respectively.

Kupczyk-Subotkowska et al. (Journal of Drug Targeting, 1997, 4(6),359–370) discloses derivatives of melphalan designed to enhance theaccumulation of melphalan into cancer cells. A number of dipeptidescomprising melphalan and valine or glutamic acid were tested and it wasconcluded that the cellular uptake of said dipeptides as well as oftheir esters probably took place via passive diffusion rather than by anamino acid or oligopeptide transporter.

One problem with the usage of bifunctional alkylating agents is primary,i.e. intrinsic, and secondary, i.e. acquired, tumour resistance to thetreatment. Attempts have been made to circumvent resistance byincreasing the dose of alkylating agent administered to the patient. Itis, however, unclear how much this increases the effective dose at thetumour cell level.

There is an urgent need for new antitumour drugs in a wide variety oftumour diseases, especially in tumours showing primary resistance toconventional therapy, and/or in tumour diseases that have developedresistance, secondary resistance, after having responded to treatmentwith conventional cancer chemotherapeutics.

SUMMARY OF THE INVENTION

The object of the invention is to provide a melphalan derivative havingan improved cytotoxic activity in human tumour cells.

The invention refers to dipeptides and tripeptides containing amelphalan unit, and one or two additional amino acids or amino acidderivatives, to the use of said peptides as medicaments, and topharmaceutical compositions comprising the peptides of the invention foruse as medicaments, especially for the treatment of various malignanttumours.

The melphalan derivatives of the invention exhibit an increased efficacyon a variety of tumour types. The hypothesis of the inventors is thatthe tumour cell uptake and intracellular accumulation of the peptides ofthe invention will be increased.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical formulas of the known compounds melphalan,sarcolysine and P2, as well as the formulas of the compuonds of theinvention J1, J3, and JV28.

FIG. 2 shows a comparison of IC50s for melphalan, J1 and P2 in 27 humantumour samples.

DESCRIPTION OF THE INVENTION

The present invention relates to new peptides of phenylalanine mustardcomprising p-[bis(2-chloroethyl)amino]-L-phenylalanine mustard, L-PAM,and p-[bis(2-chloroethyl)-amino]-D-phenylalanine mustard, D-PAM,covalently linked to one or two amino acids or amino acid derivativesforming alkylating di- or tripeptides. The L-forms are, however,preferred.

The present invention refers to new melphalan derivatives, andespecially to a di- or tripeptide having the formula I

-   wherein R₁ is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂,    alkylamino, cycloalkylamino or arylamino;-   R₂ is-   wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl,    NH-acyl, N(CH₂CH₂Cl)₂, NO₂, F, CF₃ or H, and n is 1 or 2;-   X is-   wherein R₅ is H;-   R₄ is a natural or modified cyclic or aromatic amino acid, or H; as    well as pharmaceutically acceptable salts thereof.

In said formulas alkyl preferably is a lower alkyl, that is alkyl having1–4 carbon atoms.

If n in the formula I is 1, the substituent R₃ can be in ortho-, meta-or para-position. If n is 2, the two substituents R₃ can be the same ordifferent.

Natural amino acids refers to amino acids that are normally existing andexerting their functions in living organisms. Modified amino acidsrefers to amino acids that in some way have been modified into adifferent chemical structure and chemical composition than a naturalamino acid. An example of a natural cyclic amino acid is proline.Examples of aromatic amino acids are phenylalanine, tyrosine,tryptophan, and histidine.

The N-terminus of the melphalan molecule should preferably not beprotected as amide or carbamate, this means that R₄ should preferablynot be a protecting group, such as formyl, acetyl or propionyl, orbenzoyl, as the protected form of the compound in general has a lowercytotoxic activity than the corresponding free form.

Pharmaceutically acceptable salts are for instance acid addition salts,such as salts of HCl, HBr, and methane sulphonic acid.

A preferred di- or tripeptide of the invention has the formula V

-   wherein R₁ is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂,    alkylamino, cycloalkylamino or arylamino;-   R₃ is NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂Cl)₂, NO₂,    F, CF₃ or H; and-   R₄ is a natural or modified cyclic or aromatic amino acid, or H; as    well as pharmaceutically acceptable salts thereof.

A specially interesting group of peptides in accordance with theinvention are peptides of the formula I or V, wherein R₃ is F.

Dipeptides in accordance with the invention are peptides of the formulaI or V, wherein R₁ is alkyloxy; R₃ is F, CF₃, H, OH, O-alkyl, NO₂,N(CH₂CH₂Cl)₂, NH-acyl or NH₂; and R₄ is H.

Examples of preferred dipeptides areL-melphalanyl-p-L-fluoro-phenylalanine ethyl ester (J1),L-melphalanyl-p-L-fluoro-phenylalanine isopropyl ester (JV28), andpharmaceutically acceptable salts thereof.

Tripeptides in accordance with the invention are peptides of the formulaI or V, wherein R₁ is alkyloxy; R₃ is F, CF₃, H, OH, O-alkyl, NH-acyl,NO₂, N(CH₂CH₂Cl)₂ or NH₂; and R₄ is a natural or modified cyclic oraromatic amino acid.

An example of a preferred tripeptide isL-prolyl-L-melphanalyl-p-fluorophenylalanine ethyl ester (J3), andpharmaceutically acceptable salts thereof.

All dipeptide derivatives of the invention can be synthesised fromtert.butoxycarbonyl(Boc)-protected melphalan. The coupling ofBoc-melphalan to different esters or amides was performed using either(benzotriazol-1-yloxy)tripyrrolidino-phosphonium hexafluorophosphate(PyBOP)/triethylamine or 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDC)/N-methylmorpholine (NMM)/1-hydroxybenzotriazole(HOBt) as coupling reagents. Purification procedures using silica gelchromatography allowed the isolation of pure products. Removal of theBoc-group was performed in HCl saturated ethyl acetate. The dipeptidehydrochloride salts were purified by recrystallisation inethanol/diethyl ether.

In the synthesis of tripeptide derivatives Boc-protected amino acidswere coupled to the melphalan containing dipeptide derivative usingEDC/NMM/HOBt as coupling reagents. Deprotection using HCl saturatedethyl acetate followed by recrystallization (EtOH/diethyl ether orEtOAc/diethyl ether) allowed the isolation of pure tripeptidehydrochloride salts.

The invention also refers to the use of a peptide as described above asa medicament, as well as for the manufacture of a medicament fortreatment of malignant tumours.

The alkylating peptides of the invention were found to display a broadspectrum of activity with increased potency compared to melphalan andL-prolyl-L-sarcolysyl-p-L-fluorophenylalanine ethyl ester (P2) on tumourcell lines of different histologies and also on tumour cell lines thatdisplay L-PAM resistance. Moreover, the alkylating peptides were foundto be significantly more effective compared to melphalan and P2 onfreshly obtained human tumour samples of different origin, bothhematological and solid, as shown in the examples given below.

The peptides of the invention may be used as first line treatment,either alone or in combination with other drugs or concomitant orsequenced with radiotherapy, of the following tumour diseases and/orstages of the respective disease:

-   (I) Solid tumours: for operable breast cancer as neoadjuvant or    adjuvant treatment as well as for the treatment of advanced    inoperable breast cancer, for small cell lung cancer of LD (Limited    Disease) or ED (Extensive Disease) type, for operable stages of    non-small cell lung cancer as neoadjuvant treatment, for inoperable    stage III B or IV of non-small cell lung cancer, for operable    (neoadjuvant) and inoperable head and neck cancer and esophageal    cancer, for operable ovarian cancer as adjuvant treatment and for    advanced ovarian cancer, for advanced cervical cancer, for squamous    cell and basal cell carcinoma of the skin which is non-amendable to    surgery or radiotherapy and for neuroblastoma. The above mentioned    tumour types represent (1) tumours, which have shown sensitivity to    treatment with alkylating agents, but in which this conventional    therapy is not efficient enough, or (2) tumours that frequently    relapse after a first remission and at that stage show less    sensitivity to conventional therapy (resistance). Other groups of    tumours which would be reasonable to treat with the new peptides are    different stages of bladder cancer, advanced prostate cancer,    malignant melanoma, colo-rectal cancer and soft tissue sarcomas    where palliative results frequently have been obtained with    alkylating agents. Still another group of tumours to treat are the    generally drug resistant tumours like renal cancer and brain    tumours.-   (II) Hematological and lymphatic tumours: multiple myeloma, high and    low grade lymphomas, Mb.Hodgkin, chronic lymphocytic leukemia, acute    myelogenous or lymphocytic leukemia and chronic myelogenous    leukemia.

Another object of the invention is a pharmaceutical composition for thetreatment of malignant tumours, which comprises at least one peptide asdescribed above together with at least one pharmaceutically acceptablecarrier and/or excipient.

A pharmaceutical composition according to the invention can be used forthe treatment of breast cancer, lung cancer, ovarian cancer, leukemias,lymphomas and multiple myeloma.

The pharmaceutical compositions are prepared in a manner known to aperson skilled in the pharmaceutical art. The carrier or the excipientcould be a solid, semi-solid or liquid material that could serve as avehicle or medium for the active ingredient. Suitable carriers orexcipients are known in the art. The pharmaceutical composition could beadapted to parenteral, oral or topical use and could be administered tothe patient as tablets, capsules, solutions, suspensions, ointments orthe like.

For parenteral administration the peptides according to the inventioncan be incorporated into a solution or suspension. Parenteraladministration refers to the administration by injection, for instanceby intravenous, intracapsular, intrathecal, intrapleural, intratumoral,or intraperitoneal injection or intravesically. Intravenousadministration is preferred. Bone marrow may also be treated in vitro.The pharmaceutical composition should contain at least 0.001% by weightof an active peptide according to the invention, preferably 0.1–10% byweight.

The solutions or suspensions could also comprise at least one of thefollowing adjuvants: sterile diluents such as water for injection,saline, fixed oils, polyethylene glycols, glycerol, propylene glycol orother synthetic solvents, antioxidants such as ascorbic acid or sodiumbisulfite, buffers such as acetates, citrates or phosphates, and agentsfor adjustment of the tonicity such as sodium chloride or dextrose. Theparenteral preparation could be enclosed into ampoules, disposablesyringes or multiple dosage vessels made of glass or plastic.

For intravenous injection, the pharmaceutical composition of theinvention may be administered by means of two vials, whereby vial Icomprises the peptide as a hydrochloric salt, with and without a carrieror compound that increases solubility or effects the stability, and vialII comprises a mixture of propylenglycol/ethanol. The peptide will bedissolved immediately prior to the administration and mixed with 5%glucose or saline. The dose given might be in the range of 0.1 mg/kg–1mg/kg given as a short infusion.

For topical administration the peptides according to the invention couldbe incorporated into a solution, suspension, or ointment. Saidcompositions could contain at least 0.1% by weight of the activepeptide, preferably 0.1–10% by weight.

Another object of the invention is a method for the treatment ofmalignant tumours in a subject in need thereof, comprisingadministrating a pharmaceutically effective dose of a peptide describedabove. Subject refers to any mammal, preferably a human.

EXAMPLES Syntheses of Compounds

In the following examples of synthesis of melphalan derivatives of theinvention, as well as comparative compounds, all solvents were ofanalysis or synthesis grade. 1-Hydroxybenzotriazole, HOBt;N-methylmorpholine, NMM; and/or 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride, EDC; or(benzotriazol-1-yloxy)-tripyrrolidino-phosphonium hexafluorophosphate,PyBOP; and triethyl amine were used as coupling reagents. Melting pointswere measured in a Büchi Melting Point B-540 apparatus. ¹H- and ¹³CNMR-spectra were obtained on a JEOL JNM-EX 400 NMR-spectrometer.¹H-spectra were recorded at 400 MHz and ¹³C-spectra at 100 MHz,respectively. The reactions were monitored by thin-layer chromatography(TLC), on silica plated aluminum sheets (Silica gel 60 P254, E. Merck),detecting spots by UV-light and/or 2% ninhydrin in ethanol followed byheating. Column chromatography was performed on wet packed silica(Silica gel 60 (0.040–0.063 mm), E. Merck) using flash chromatography.

The L-melphalan used as a starting compound was obtained from Sigma andwas recrystallised from ethanol prior to use. The intermediateN-tert-butoxycarbonyl-L-melphalan was synthesised according to Pai, N.N.; Miyawa, J. H.; Perrin, J. H. Drug Dev. Industr. Pharm. 1996, 22,181–184. L-Melphalan (500 mg, 1.64 mmol) was dissolved in 50% aqueousTHF (5 ml) and triethylamine (201 μl, 1.44 mmol) was added. The solutionwas cooled to 0° C. and di-tert-dibutyl-dicarbonate (210 mg, 0.96 mmol)dissolved in THF (3 ml) was added dropwise. The solution was stirred for30 min at 0° C. and then for 18 h at room temperature (RT). The solventwas evaporated off and water was added and the solution was acidified topH 5 with 10% citric acid. After extraction three times with ethylacetate the combined organic extract was dried over MgSO₄. The solventwas evaporated off and the residue was purified by flash chromatographyon silica using chloroform:methanol in 3:1 and 19:1 as eluents affording48% of a pure product. ¹H NMR (CDCl₃) δ 7.05 (d, 2H, Ph-H), 6.58 (d, 2H,Ph-H), 4.98 (br s, 1 H, NH), 4.47 (br s, 1 H, α-H), 3.69–3.49 (m, 8H, 4CH₂-mustard), 3.12–2.91 (m, 2H, CH₂-Ph), 1.40 (s, 9H, CH₃-Boc).

Example 1 L-Melphalanyl-L-p-fluorophenylalanine ethyl esterhydrochloride (J1)

L-p-Fluorophenylalanine (217 mg, 1.18 mmol) was dissolved in EtOH (5 ml)previously bubbled with HCl. The reaction was brought to 100° C. and wasallowed to reflux for 18 hours. The solvent was evaporated off and theproduct was dried under high vacuum, affording L-p-fluorophenylalanineethyl ester hydrochloride as dry white crystals (98%). ¹H NMR: (CD₃OD) δ7.30–7.26 (m, 2H, Ph-H), 7.13–7.06 (m, 2H, Ph-H), 4.29–4.22 (m, 2H,CH₂-Ph), 3.31–3.10 (m, 3H, CH₂CH₃, α-H), 1.24 (t, 3H, CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalan (157 mg, 0.387 mmol) was dissolved indichloromethane (4 ml). PyBOP (201 mg, 0.387 mmol) and triethylamine (54μl, 0.387 mmol) were added and the solution was stirred at roomtemperature for 1 h. A solution of L-p-fluorophenylalanine ethyl esterhydrochloride (92 mg, 0.387 mmol) and triethylamine (54 μl, 0.387 mmol)in dichloromethane (4 ml) was added and the reaction was stirred overnight. The reaction was stopped by extraction with saturated NaHCO₃followed by 10% citric acid. The organic layer was dried by MgSO₄ andthe solvent evaporated to give 200 mg of a yellow oil which was purifiedby gradient column chromatography using ether:pentane (1:2, 1:1, 1:0) aseluent to afford 102 mg (43% yield) ofN-tert-butoxycarbonyl-L-melphalanyl-L-p-fluorophenylalanine ethyl esterwhich was used in the next step-without further purification.

N-tert-Butoxycarbonyl-L-melphalanyl-L-p-fluorophenylalanine ethyl ester(64 mg, 0.11 mmol) was dissolved in 5 ml EtOAc previously bubbled withHCl (gas). The mixture was stirred at RT for 30 minutes. The solvent wasremoved in vacuo. The residue was recrystallized from EtOH/Et₂O.L-Melphalanyl-L-p-fluorophenylalanine ethyl ester hydrochloride (J1) wasisolated as white crystals.

¹H NMR: (CD₃OD) δ 7.23 (d, 2H, Ph-H, Phe), 7.13 (d, 2H, Ph-H, Phe), 7.01(d, 2H, Ph-H, Mel), 6.72 (d, 2H, Ph-H, Mel), 4.68 (dd, 1H, α-CH, Phe),4.61 (br s, 1H, α-CH, Mel), 4.12 (q, 2H, CH₂CH₃), 3.80–3.62 (m, 8H,CH₂-mustard), 3.22–2.86 (m, 4H, CH₂-Ph), 1.21 (t, 3H, CH₂CH₃). Elementalanalysis CHN 53.7;5.8;7.9 (calculated 54.1;6.1;7.9)

Example 2 L-Prolinyl-L-melphalanyl-L-p-fluorophenylalanine ethyl esterhydrochloride (J3)

N-tert-Butoxycarbonyl-L-proline (12 mg, 0.054 mmol),L-melphalanyl-L-p-fluorophenylalanine ethyl ester hydrochloride (24 mg,0.045 mmol), HOBt (8 mg, 0.054 mmol), and NMM (7 μl, 0.045 mmol) weredissolved in dichloromethane (5 ml). The solution was cooled to 0° C.and EDC hydrochloride (11 mg, 0.045 mmol) was added. The solution wasstirred for 1 h at 0° C. and then overnight at RT. The reaction mixturewas diluted to 10 ml with dichloromethane and the reaction stopped bysuccessive extractions with 10% aqueous citric acid, saturated NaHCO₃and brine. The organic phase was dried over anhydrous Na₂SO₄ and thesolvent evaporated off under reduced pressure to afford 27 mg (86%)N-tert-butoxycarbonyl-L-prolinyl-L-melphalanyl-L-p-fluorophenylalanineethyl ester that was used in the next step without further purification.

¹H NMR (CDCl₃) δ 7.08–6.89 (m, 6H, Ph-H, Phe, Mel), 6.67 (br s, 1H, NH),6.55 (d, 2H, Ph-H), 6.24 (br s, 1H, NH), 4.72 (m, 1H, α-CH), 4.55 (br s,1H, α-CH), 4.20 (br s, 1H, α-CH), 4.10 (q, 2H, CH₂CH₃), 3.74–3.54 (m,8H, CH₂-mustard), 3.42–3.20 (m, 2H, proline δ), 3.10–2.86 (m, 4H,CH₂-Ph), 2.20–1.74 (m, 4H, proline β,γ), 1.40 (s, 9H, CH₃-Boc), 1.18 (t,3H, CH₂CH₃).

N-tert-Butoxycarbonyl-L-prolinyl-L-melphalanyl-L-p-fluorophenylalanineethyl ester (25 mg) was dissolved in HCl saturated ethyl acetate (3 ml).The mixture was stirred at RT for 30 minutes. The solvent was removed invacuo. Recrystallization from ethyl acetate and diethyl ether yieldedpure J3 as off-white crystals in 94% yield (calculated fromL-melphalanyl-L-p-fluorophenylalanine ethyl ester hydrochloride).

¹H NMR (CD₃OD) δ 7.26–6.94 (m, 6H, Ph-H, Phe, Mel), 6.68 (d, 2H, Ph-H),6.24 (br s, 1H, NH), 4.62–4.55 (m, 2H, α-CH), 4.20–4.05 (m, 3H, α-CH,CH₂CH₃), 3.74–3.54 (m, 8H, CH₂-mustard), 3.25–2.75 (m, 6H, CH₂-Ph,proline δ), 2.40–1.85 (m, 4H, proline β,γ), 1.23 (t, 3H, CH₂CH₃).

Example 3 L-Melphalanyl-L-phenylalanine ethyl ester hydrochloride (JV22)

N-tert-Butoxycarbonyl-L-melphalan (150 mg; 0.37 mmol) was dissolved in2.6 ml dichloromethane. PyBOP (199 mg; 0.38 mmol) and triethylamine (104μl; 0.75 mmol) were added. After stirring at RT for 30 minutes asolution of triethylamine (104 μl; 0.75 mmol) and phenylalanine ethylester hydrochloride (89 mg; 0.39 mmol) in 2.6 ml dichloromethane wasadded. After 4 h at RT the reaction was quenched. Dichloromethane wasadded up to a total volume of 20 ml before extraction with 20 mlsaturated aqueous NaHCO₃ and 20 ml 10% citric acid. The organic layerwas dried (MgSO₄), filtered and concentrated in vacuo. The crude productwas purified by column chromatography on silica using a gradient ofether:pentane (2:1→3:1), followed by ether and thereafter by CHCl₃:MeOH(19:1) as eluents. Collection and concentration of adequate fractionsgave pure N-tert-butoxycarbonyl-L-melphalanyl-L-phenylalanine ethylester as a white solid (110 mg, 51%). Mp: 117–120° C.

¹H NMR (CDCl₃) δ 7.29–7.16 (m, 3H, Ph-H, Phe), 7.06–6.95 (m, 4H, Ph-H),6.57 (d, 2H, Ph-H Mel), 6.28 (br s, 1H, NH, Phe), 4.92 (br s, 1H, NH,Mel), 4.76 (br s, 1H, α-CH, Phe); 4.26 (br s, 1H, α-CH, Mel), 4.10 (q,2H, CH₂CH₃), 3.74–3.50 (m, 8H, CH₂-mustard), 3.12–2.84 (m, 4H, CH₂-Ph),1.40 (s, 9H, CH₃-Boc), 1.18 (t, 3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.05 (2C:s) (C═O amide and ester), 155.10 (C═O, Boc), 145.10 (C-4′), 135.84(Ph), 130.75 (2 C:s), 129.39 (2 C:s), 128.54 (2 C:s) (Ph and C-3′),127.13 (Ph), 125.07 (C-1′), 112.27 (2 C:s) (C-2′), 79.82 (C-Boc), 61.54(CH₂CH₃), 55.51 (α-CH), 53.55 (2 C:s) (N—CH₂), 53.32 (α-CH), 40.51 (2C:s) (CH₂—Cl), 38.13, 36.73 (CH₂-Ph), 28.35 (3 C:s) (CH₃-Boc), 14.17(CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalanyl-L-phenylalanine ethyl ester (100 mg;0.17 mmol) was dissolved in 6 ml HCl saturated ethyl acetate. Themixture was stirred at RT for 30 minutes. The solvent was removed invacuo. Recrystallization from ethyl acetate and diethyl ether yieldedpure JV22 (37 mg; 42%) as a slightly yellowish/brownish solid. Mp:123–125° C.

¹H NMR (CDCl₃) δ 7.31–7.13 (m, 7H, Ph-H), 6.72 (d, 2H, Ph-H, Mel),4.73–4.66 (m, 1H, α-CH-Phe), 4.12 (q, 2H, CH₂CH₃), 3.99–3.92 (m, 1H,α-CH, Mel), 3.77–3.64 (m, 8H, CH₂-mustard), 3.20–2.85 (m, 4H, CH₂-Ph),1.18 (t, 3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.05, 168.42 (C═O in amide andester), 145.96 (C-4′), 136.55 (Ph), 130.43 (2 C:s) (C-3′), 128.90 (2C:s), 128.27 (2 C:s), 126.10 (Ph), 122.35 (C-1′), 112.58 (2 C:s) (C-2′),61.23 (CH₂CH₃), 54.29, 54.25 (α-CH), 53.06 (2 C:s) (N—CH₂), 40.26 (2C:s) (CH₂—Cl), 37.12, 36.29 (CH₂-Ph), 13.10 (CH₂CH₃).

Example 4 L-Melphalanyl-L-tyrosine ethyl ester hydrochloride (JV24)

N-tert-Butoxycarbonyl-L-melphalan (150 mg; 0.37 mmol) was dissolved indichloromethane (3 ml). PyBOP (199 mg; 0.38 mmol) and triethylamine (104μl; 0.75 mmol) were added. After stirring at RT for 30 minutes asolution of triethylamine (104 μl ; 0.75 mmol) and tyrosine ethyl esterhydrochloride (94 mg; 0.38 mmol) in 3 ml dichloromethane was added.After 100 min stirring at RT the reaction was quenched. Dichloromethanewas added up to a total volume of 20 ml before extraction with 20 mlsaturated aqueous NaHCO₃ and 20 ml 10% citric acid. The organic layerwas dried (MgSO₄), filtered and concentrated in vacuo. The crude productwas purified four times by column chromatography on silica using agradient eluent systems of ether:pentane (2:1→4:1→1:0) followed byCHCl₃:MeOH (19:1→97:3). PureN-tert-butoxycarbonyl-L-melphalanyl-L-tyrosine ethyl ester was isolatedas a colorless oil (59 mg; 27%).

¹H NMR (CDCl₃) δ 7.05–6.92 (m, 2H, Ph-H-Mel), 6.86–6.74 (m, 2H,Ph-H-Phe), 6.67 (d, 2H, Ph-H, Phe), 6.59–6.43 (m, 2H, Ph-H, Mel),6.42–6.36 (m, 1H, NH, Phe), 5.00 (br s, 1H, NH, Mel), 4.81–4.70 (m, 1H,α-CH, Phe), 4.26 (br s, 1H, α-CH, Mel), 4.12 (q, 2H, CH₂CH₃), 3.70–3.53(m, 8H, CH₂-mustard), 3.03–2.85 (m, 4H, CH₂-Ph), 1.39 (s, 9H, CH₃-Boc),1.21 (t, 3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.21 (2 C:s) (C═O in amide andester), 155.31 (2 C:s) (C═O Boc and C-4″), 144.96 (C-4′), 130.70 (2C:s), 130.47 (2 C:s) (Ph and C-3′), 127.12 (Ph), 125.21 (C-1′), 115.52(2 C:s) (Ph), 112.31 (2 C:s) (C-2′), 80.09 (C-Boc), 61.61 (CH₂CH₃),55.84 (α-CH), 53.56 (2 C:s) (N—CH₂), 53.25 (α-CH), 40.55, 40.50(CH₂—Cl), 37.32 (2 C:s) (CH₂-Ph), 28.36 (3 C:s) (CH₃-Boc), 14.22(CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalanyl-L-tyrosine ethyl ester (48 mg; 81μmol) was dissolved in 4 ml HCl saturated ethyl acetate. The mixture wasstirred at RT for 30 minutes. The solvent was removed in vacuo.Recrystallization from ethyl acetate and diethyl ether yielded the pureJV24 (13 mg; 30%) as a slightly brownish solid. Mp: 150–154° C.

¹H NMR (CDCl₃) δ 7.16–6.86 (m, 4H, Ph-H), 6.75–6.65 (m, 4H, Ph-H),4.68–4.59 (m, 1H, α-CH, Phe), 4.13 (q, 2H, CH₂CH₃), 4.02–3.94 (m, 1H,α-CH, Mel), 3.77–3.60 (m, 8H, CH₂-mustard), 3.21–2.71 (m, 4H, CH₂-Ph),1.20 (t, 3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 170.94, 168.08 (C═O in amide andester), 156.12 (C-4″), 145.68 (C-4′), 130.41 (2 C:s), 129.68(2 C:s),127.03, (Ph and C-3′), 125.86 (C-1′), 114.98 (2 C:s) (Ph), 112.34 (2C:s) (C-2′), 60.80 (CH₂CH₃), 54.48, 54.27 (α-CH), 53.07 (2 C:s) (N—CH₂),40.19 (2 C:s) (CH₂—Cl), 36.32 (2 C:s) (CH₂-Ph), 12.64 (CH₂CH₃).

Example 5 L-Melphalanyl-L-p-methoxyphenylalanine ethyl esterhydrochloride (JV25)

N-tert-Butoxycarbonyl-L-tyrosine ethyl ester (228 mg; 0.74 mmol) wasdissolved in 15 ml acetone, and K₂CO₃ (123 mg, 0.89 mmol) was added.Dimethyl sulfate (77.5 μl; 0.81 mmol) was carefully added and thesolution was refluxed for 20 h. Solid K₂CO₃ was removed by filtrationand the acetone was removed in vacuo. Purification by columnchromatography on silica using CHCl₃:MeOH:heptane (4:1:5) as eluentyielded 214 mg (90%) of N-tert-butoxycarbonyl-L-p-methoxyphenylalanineethyl ester as a clear oil. This intermediate was used in the next stepwithout further characterization.

N-tert-Butoxycarbonyl-L-p-methoxyphenylalanine ethyl ester (165 mg; 0.51mmol) was dissolved in CHCl₃ (8.5 ml) and iodotrimethylsilane (168μ;1.22 mmol) was added. The solution was stirred for 40 min at RT. Thereaction was quenched by addition of MeOH (175 μl; 4.3 mmol). Thesolvent and excess iodotrimethylsilane were removed in vacuo to yield169 mg (149%) of the crude product (L-p-methoxyphenylalanine ethylester) as a brown-orange solid which was used in the next step withoutfurther purification. ¹H NMR (CD₃OD) δ 7.16 (d, 2H, Ph-H), 6.90 (d, 2H,Ph-H), 4.28–4.18 (m, 3H, CH₂CH₃, α-CH), 3.78 (s, 3H, OCH₃), 3.21–3.08(m, 2H, CH₂-Ph), 1.24 (t, 3H, CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalan (150 mg; 0.37 mmol) was dissolved in 3ml dichloromethane. PyBOP (199 mg; 0.38 mmol) and triethylamine (104 μl;0.75 mmol) were added. After stirring at RT for 30 minutes a solution oftriethylamine (104 μL; 0.75 mmol) and L-p-methoxyphenylalanine (124 mg;0.55 mmol) in 3 ml dichloromethane was added. The solution was stirredat RT over night. Dichloromethane was added up to a total volume of 20ml before extraction with 20 ml saturated aqueous NaHCO₃ and 20 ml 10%citric acid. The organic layer was dried (MgSO₄), filtered andconcentrated in vacuo. The crude product was purified four times byflash column chromatography on silica, twice with ether:heptane(3:1),and twice with CHCl₃:MeOH (19:1) as eluents, to yieldN-tert-butoxycarbonyl-L-melphalanyl-L-p-methoxyphenylalanine ethyl esteras a white solid (150 mg; 66%). Mp: 125–127.5° C.

¹H NMR (CDCl₃) δ 7.06 (d, 2H, Ph-H, Mel), 6.90 (d, 2H, Ph-H, Phe), 6.76(d, 2H, Ph-H, Phe), 6.59 (d, 2H, Ph-H, Mel), 6.31 (br s, 1H, NH, Phe),4.97 (br s, 1H, NH, Mel), 4.71 (br s, 1H, α-CH, Phe), 4.27 (br s, 1H,α-CH, Mel), 4.10 (q, 2H, CH₂CH₃), 3.75 (s, 3H, OCH₃), 3.69–3.56 (m, 8H,CH₂-mustard), 3.00–2.85 (m, 4H, CH₂-Ph), 1.40 (s, 9H, CH₃-Boc), 1.19 (t,3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.11, 170.91 (C═O in amide and ester),158.72 (C-4″), 155.30 (C═O, Boc), 145.03 (C-4′), 130.76 (2 C:s), 130.39(2 C:s), (Ph and C-3′), 127.75 (Ph), 125.36 (C-1′); 113.96 (2 C:s) (Ph),112.28 (2 C:s) (C-2′), 80.12 (C-Boc), 61.50 (CH₂CH₃), 55.81 (α-CH),55.27 (OCH₃) 53.55 (2 C:s) (N—CH₂), 53.46 (α-CH), 40.52 (2 C:s)(CH₂—Cl), 37.33, 37.23 (CH₂-Ph), 28.34 (3 C:s) (CH₃-Boc), 14.20(CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalanyl-L-p-methoxyphenylalanine ethyl ester(140 mg; 0.23 mmol) was dissolved in 17 ml HCl saturated ethyl acetate.The mixture was stirred at RT for 3 h. The solvent was removed in vacuo.The crude product was dissolved in ethanol followed by precipitation indiethyl ether affording JV25 (45 mg; 36%) as a white solid. Mp: 170–173°C.

¹H NMR (CDCl₃) δ 7.17–7.07 (m, 4H, Ph-H), 6.83 (d, 2H, Ph-H, Phe), 6.72(d, 2H, Ph-H, Mel), 4.68–4.61 (m, 1H, α-CH, Phe), 4.13 (q, 2H, CH₂CH₃),3.99–3.94 (m, 1H, α-CH, Mel), 3.77–3.59 (m, 11H, OCH₃ and CH₂-mustard),3.17–2.82 (m, 4H, CH₂-Ph), 1.19 (t, 3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ171.11, 168.41 (C═O in amide and ester), 158.91 (C-4″), 146.10 (C-4′),130.44 (2 C:s), 129.94 (2 C:s) (Ph and C-3′), 128.38 (Ph), 122.18(C-1′), 113.67 (2 C:s) (Ph), 112.18 (2 C:s) (C-2′), 61.19 (CH₂CH₃),54.45, 54.37, 54.29 (α-CH and OCH₃), 52.99 (2 C:s) (N—CH₂), 40.31 (2C:s) (CH₂—Cl), 36.34, 36.29 (CH₂-Ph), 13.14 (CH₂CH₃).

Example 6 L-Melphalanyl-L-p-nitrophenylalanine ethyl ester hydrochloride(JV26)

N-tert-Butoxycarbonyl-L-p-nitrophenylalanine (395 mg; 1.27 mmol) wasdissolved in HCl saturated ethanol (20 ml). The solution was heated atreflux for 20 h. The mixture was partitioned between CHCl₃ and 1M HCl(pH 4). The aqueous layer was basified with 5% KOH to pH 10 and was thenextracted four times with CHCl₃. The organic layers were combined, dried(MgSO₄), filtered, and concentrated in vacuo to affordL-p-nitrophenylalanine ethyl ester as a thin yellowish oil (195 mg; 65%)that was used in the next step without further purification.

¹H NMR (CDCl₃) δ 8.14 (d, 2H, Ph-H), 7.37 (d, 2H, Ph-H), 4.14 (q, 2H,CH₂CH₃), 3.71 (t, 1H, α-CH), 3.16–2.91 (m, 2H, CH₂-Ph), 1.22 (t, 3H,CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalan (86 mg; 0.21 mmol) was dissolved indichloromethane (3 ml). PyBOP (115 mg; 0.22 mmol) and triethylamine (58μl; 0.42 mmol) were added. After stirring at RT for 30 minutes asolution of triethylamine (29 μl; 0.21 mmol) and L-p-nitrophenylalanineethyl ester (55 mg; 0.23 mmol) in 3 ml dichloromethane was added. Thesolution was stirred at RT over night. Dichloromethane was added up to atotal volume of 10 ml before extraction with 10 mL saturated aqueousNaHCO₃ and 10 ml 10% citric acid. The organic layer was dried (MgSO₄),filtered and concentrated in vacuo. The crude product was purified twiceby flash column chromatography on silica using CHCl₃:MeOH (9:1 and 19:1)as eluent, to yield pureN-tert-butoxycarbonyl-L-melphalanyl-L-p-nitrophenylalanine ethyl esteras a light yellow solid (90 mg; 69%). Mp 152–155° C. ¹NMR (CDCl₃) δ8.09–8.02 (m, 2H, Ph-H, Phe), 7.21–7.01 (m, 4H, Ph-H), 6.57 (d, 2H,Ph-H, Mel), 6.50–6.44 (m, 1H, NH, Ph), 4.92 (br s, 1H, NH, Mel), 4.77(br s, 1H, α-CH, Phe), 4.25 (br s, 1H, α-CH, Mel), 4.11 (q, 2H, CH₂CH₃),3.70–3.53 (m, 8H, CH₂-mustard), 3.25–3.10 (m, 2H, CH₂-Ph, Phe),2.97–2.84 (m, 2H, CH₂-Ph, Mel) 1.40 (s, 9H, CH₃-Boc), 1.19 (t, 3H,CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.31, 170.40 (C═O in amide and ester),155.46 (C═O, Boc), 147.14 (Ph), 145.10 (C-4′), 143.85 (Ph), 130.68 (2C:s), 130.41(2 C:s) (Ph and C-3′), 125.21 (C-1′), 123.62 (2 C:s) (Ph),112.40 (2 C:s) (C-2′), 80.45 (C-Boc), 61.95 (CH₂CH₃), 55.98 (α-C), 53.54(2 C:s) (N—CH₂), 53.03 (α-CH), 40.46 (2 C:s) (CH₂—Cl), 37.86, 37.00(CH₂-Ph), 28.33 (3 C:s) (CH₃-Boc), 14.20 (CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalanyl-L-p-nitro-phenylalanine ethyl ester(80 mg; 0.13 mmol) was dissolved in 6 ml HCl saturated ethyl acetate.The mixture was stirred at RT for 1.5 h. The solvent was removed invacuo. The crude product was dissolved in ethanol followed byprecipitation in diethyl ether to afford JV26 (55 mg; 76%) as a lightorange solid. Mp: 138–142° C.

¹H NMR (CDCl₃) δ 8.20–8.06 (m, 2H, Ph-H, Phe), 7.52–7.33 (m, 2H, Ph-H,Phe), 7.17–6.85 (m, 2H, Ph-H, Mel), 6.77–6.60 (m, 2H, Ph-H, Mel), 4.16(q, 2H, CH₂CH₃), 4.00–3.94 (m, 1H, α-CH, Mel), 3.79–3.54 (m, 8H,CH₂-mustard), 3.19–2.71 (m, 4H, CH₂-Ph), 1.21 (t, 3H, CH₂CH₃). ¹³C NMR(CDCl₃) δ 170.46, 168.29 (C═O in amide and ester), 147.16 (Ph), 145.61(C-4′), 144.67 (Ph), 130.96 (2 C:s), 130.24 (2 C:s) (Ph and C-3′),126.72 (C-1′), 123.26 (2 C:s) (Ph), 115.45 (2 C:s) (C-2′), 61.53(CH₂CH₃), 54.76, 54.08 (α-CH), 53.54 (2 C:s) (N—CH₄), 39.22 (2 C:s)(CH₂—Cl), 36.75, 36.33 (CH₂-Ph), 13.16 (CH₂CH₃).

Example 7 L-Melphalanyl-L-melphalan ethyl ester hydrochloride (JV27)

N-tert-Butoxycarbonyl-L-melphalan (35 mg; 86 μmol) was dissolved indichloromethane (2 ml). PyBOP (43 mg; 83 μmol) and triethylamine (23 μl;162 μmol) were added. After stirring at RT for 30 minutes a solution oftriethylamine (23 μl; 162 μmol) and L-melphalan ethyl esterhydrochloride (32 mg; 87 μmol) in 2 ml dichloromethane was added. Thesolution was stirred at RT over night, and more dichloromethane (up to10 ml) was added before extraction with 10 ml saturated aqueous NaHCO₃and 10 ml 10% citric acid. The organic layer was dried (MgSO₄), filteredand concentrated in vacuo. Purification by column chromatography onsilica using CHCl₃:MeOH:heptane (4:1:7) and CHCl₃:MeOH (19:1) as eluentsgave pure N-tert-butoxycarbonyl-L-melphalanyl-L-melphalan-ethyl ester asa brown gum (29 mg; 49%).

¹H NMR (CDCl₃) δ 7.06 (d, 2H, Ph-H), 6.87 (d, 2H, Ph-H), 6.61–6.46 (m,4H, Ph-H), 6.24 (br s, 1H, NH), 4.99 (br s, 1H, NH), 4.68 (br s, 1H,α-CH), 4.25 (br s, 1H, α-CH), 4.12 (q, 2H, CH₂CH₃), 3.72–3.47 (m, 16H,CH₂-mustard), 3.02–2.87 (m, 4H, CH₂-Ph), 1.40 (s, 9H, CH₃-Boc), 1.21 (t,3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.16, 170.71 (C═O in amide and ester),154.87 (C═O, Boc), 145.19 (2 C:s) (C-4′ and C-4″), 130.79 (2 C:s),130.68 (2 C:s) (C-3′ and C-3″), 125.08, 124.33 (C-1′, C-1″), 112.30 (2C:s), 112.04 (2 C:s) (C-2′ and C-2″), 79.65 (C-Boc), 61.50 (CH₂CH₃),55.83 (α-CH), 53.57 (2 C:s) (N—CH₂), 53.53 (α-CH), 40.55, 40.47(CH₂—Cl), 37.04, 36.98 (CH₂-Ph), 28.36 (3 C:s) (CH₃-Boc), 14.24(CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalanyl-L-melphalan ethyl ester (20 mg; 28μmol) was dissolved in 3 ml HCl saturated ethyl acetate. The mixture wasstirred at RT for 1.5 h. The solvent was removed in vacuo. The crudeproduct was dissolved in ethanol followed by precipitation in diethylether to afford JV27 (11 mg; 62%) as a light brown solid.

Mp: 157–160° C. ¹H NMR (CDCl₃) δ 7.19–7.01 (m, 4H, Ph-H), 6.80–6.63 (m,4H, Ph-H), 4.66–4.61 (m, 1H, α-CH), 4.13 (q, 2H, CH₂CH₃), 4.02–3.96 (m,1H, α-CH), 3.83–3.54 (m, 16H, 8 CH₂-mustard), 3.18–2.81 (m, 4H, CH₂-Ph),1.17 (t, 3H, CH₂CH₃).

Example 8 L-Melphalanyl-L-p-fluorophenylalanine isopropyl esterhydrochloride (JV28)

L-p-Fluorophenylalanine (266 mg, 1.45 mmol) was dissolved in HClsaturated isopropanol (10 ml). The solution was heated at reflux for 2.5h. The solvent was evaporated to yield the product as a white cottonlikesolid (350 mg, 91%). The product was used in the next step withoutfurther purification. Mp 226–229° C.

¹H NMR (CD₃OD) δ 7.31–7.23 (m, 2H, Ph-H), 7.12–7.03 (m, 2H, Ph-H),5.10–5.00 (m, 1H, CH-isopropyl), 4.22 (t, 1H, α-CH), 3.22–3.11 (m, 2H,CH₂-Ph), 1.25 (d, 3H, CH₃-isopropyl), 1.18 (d, 3H, CH₃-isopropyl).

N-tert-Butoxycarbonyl-L-melphalan (52 mg; 0.13 mmol) was dissolved indichloromethane (2 ml). PyBOP (71 mg, 0.14 mmol) and triethylamine (38μl; 0.27 mmol) were added. After stirring at RT for 30 minutes asolution of triethylamine (38 μl; 0.27 mmol) and L-p-fluorophenylalanineisopropyl ester (39 mg; 0.15 mmol) in 2 ml dichloromethane was added.The solution was stirred at RT over night. Dichloromethane was added upto a total volume of 10 ml before extraction with 10 ml saturatedaqueous NaHCO₃ and 10 m 10% citric acid. The organic layer was dried(MgSO₄), filtered and concentrated in vacuo. The crude product waspurified by flash column chromatography on silica using CHCl₃:MeOH(19:1) as eluent, to yield pureN-tert-butoxycarbonyl-L-melphalanyl-L-p-fluorophenylalanine isopropylester as a weakly orange semisolid (57 mg; 71%).

¹H NMR (CDCl₃) δ 7.06–6.85 (m, 6H, Ph-H), 6.56 (d, 2H, Ph-H, Mel), 6.40(br s, 1H, NH, Phe), 5.00–4.85 (m, 2H, CH-isopropyl, NH, Mel), 4.68 (brs, 1H, α-CH, Phe), 4.27 (br s, 1H, α-CH, Mel), 3.69–3.52 (m, 8H,CH₂-mustard), 3.02–2.85 (m, 4H, CH₂-Ph), 1.37 (s, 9H, CH₃-Boc), 1.18 (d,3H, CH₃-isopropyl), 1.14 (d, 3H, CH₃-isopropyl). ¹³C NMR (CDCl₃) δ171.00, 170.43 (C═O in amide and ester), 161.95 (d, J=245.0 Hz, C-4″);155.35 (C═O, Boc), 145.12 (C-4′), 131.60 (C-1″), 130.99 (2 C:s) (d,J=7.75 Hz, C-2″), 130.72 (2 C:s) (C-3′), 125.26 (C-1′), 115.29 (2 C:s)(d, J=20.6 Hz, C-3″), 112.27 (2 C:s) (C-2′), 80.24 (C-Boc), 69.52(CH-isopropyl), 55.82 (α-CH), 53.53 (2 C:s) (N—CH₂), 53.40 (α-CH), 40.48(2 C:s) (CH₂—Cl), 37.29 (2 C:s) (CH₂-Ph), 28.33 (3 C:s) (CH₃-Boc),21.81, 21.73 (CH₃-isopropyl).

N-tert-Butoxycarbonyl-L-melphalanyl-L-p-fluoro-phenylalanine isopropylester (48 mg; 79 μmol) was dissolved in 4 ml HCl saturated ethylacetate. The mixture was stirred at RT for 30 minutes. The solvent wasremoved in vacuo. The crude product was dissolved in ethanol followed byprecipitation in diethyl ether to afford JV28 (22 mg; 50%) as a whitesolid. Mp: 192–195° C.

¹H NMR (CDCl₃) δ 7.28–7.17 (m, 2H, Ph-H, Phe), 7.14 (d, 2H, Ph-H, Phe),7.04–6.96 (m, 2H, Ph-H, Mel), 6.76–6.67 (m, 2H, Ph-H, Mel), 4.68–4.60(m, 1H, α-CH, Phe), 4.00–3.93 (m, 1H, α-CH, Mel), 3.76–3.58 (m, 8H,CH₂-mustard), 3.18–2.80 (m, 4H, CH₂-Ph), 1.24 (d, 3H, CH₃-isopropyl),1.15 (d, 3H, CH₃-isopropyl). ¹³C NMR (CDCl₃) δ 170.49, 168.42 (C═O inamide and ester), 162.06 (d, J=242.4 Hz, C-4″), 146.13 (C-4′), 132.48(C-1″), 130.72 (2 C:s) (d, J=7.64 Hz, C-2″), 130.40 (2 C:s) (C-3′),122.10 (C-1′), 114.87 (2 C:s) (d, J=21.2 Hz, C-3″), 112.45 (2 C:s)(C-2′), 69.27 (CH-isopropyl), 54.28 (2 C:s) (α-CH), 52.97 (2 C:s)(N—CH₂), 40.26 (2 C:s) (CH₂—Cl), 36.31 (2 C:s) (CH₂-Ph), 20.70, 20.56(CH₃-isopropyl).

Example 9 L-Melphalanyl-L-p-aminophenylalanine ethyl ester hydrochloride(JV29)

N-tert-Butoxycarbonyl-L-p-nitrophenylalanine (510 mg; 1.64 mmol) wasdissolved in 80% ethanol (12.5 ml). A solution of CaCl₂ (150 mg; 1.35mmol) in water (1 ml) was added together with powdered zinc (3.79 g; 58mmol). After heating at reflux for 3.5 h the suspension was filtered andthe zinc powder was rinsed with excess ethanol. The filtrate wasconcentrated in vacuo to afford 600 mg (130%) ofN-tert-butoxycarbonyl-L-p-aminophenylalanine as a light yellow solidwhich still contained some ethanol. The product was used in the nextstep without further purification.

¹H NMR (CD₃OD) δ 6.96 (d, 2H, Ph-H), 6.63 (d, 2H, Ph-H), 4.16–4.07 (m,1H, α-CH), 3.05–2.76 (m, 2H, CH₂-Ph), 1.38 (s, 9H, CH₃-Boc).

N-tert-Butoxycarbonyl-L-p-aminophenylalanine (201 mg; 0.72 mmol) wasdissolved in HCl saturated ethanol (10 ml). The solution was heated atreflux for 3 h. The mixture was partitioned between CHCl₃ and 1M HCl (pH4). The aqueous layer was basified with 5% KOH to pH 10 and was thenextracted four times with CHCl₃. The organic layers were combined, dried(MgSO₄), filtered, and concentrated in vacuo to affordL-p-aminophenylalanine ethyl ester as a yellowish oil (90 mg; 60%) whichwas used in the next step without further purification.

¹H NMR (CDCl₃) δ 6.97 (d, 2H, Ph-H), 6.61 (d, 2H, Ph-H), 4.13 (q, 2H,CH₂CH₃), 3.71 (t, 1H, α-CH), 3.04–2.78 (m, 2H, CH₂-Ph), 1.23 (t, 3H,CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalan, (44 mg; 0.11 mmol) was dissolved indichloromethane (2 ml). PyBOP (55 mg; 0.11 mmol) and triethylamine (28μl; 0.20 mmol) were added. After stirring at RT for 30 minutes asolution of triethylamine (14 μl; 0.10 mmol) and L-p-aminophenylalanineethyl ester (21 mg; 0.10 mmol) in 2 ml dichloromethane was added. Thesolution was stirred at RT over night. Dichloromethane was added up to atotal volume of 10 ml before extraction with 10 ml saturated aqueousNaHCO₃ and 10 ml 10% citric acid. The organic layer was dried (MgSO₄),filtered and concentrated in vacuo. The crude product was purified twiceby flash column chromatography on silica using CHCl₃:MeOH (9:1and 19:1)as eluents, to affordN-tert-butoxycarbonyl-L-melphalanyl-L-p-aminophenylalanine ethyl esteras a brown-orange oil (28 mg; 47%).

¹H NMR (CDCl₃) δ 7.03 (d, 2H, Ph-H, Mel), 6.75 (d, 2H, Ph-H; Phe),6.59–6.52 (m, 4H, Ph-H), 6.26 (br s, 1H, NH, Phe), 4.96 (br s, 1H, NH,Mel), 4.68 (br s, 1H, α-CH, Phe), 4.26 (br s, 1H, α-CH, Mel), 4.10 (q,2H, CH₂CH₃), 3.70–3.53 (m, 8H, CH₂-mustard), 2.97–2.88 (m, 4H, CH₂-Ph),1.38 (s, 9H, CH₃-Boc), 1.19 (t, 3H, CH₂CH₃). ¹³C NMR (CDCl₃) δ 171.21,170.83 (C═O in amide and ester), 145.37, 144.96 (C-4′, C-4″), 130.78 (3C:s), 130.21 (2 C:s) (Ph and C-3′), 125.37 (C-1′), 115.22 (2 C:s) (Ph),112.26 (2 C:s) (C-2′), 80.09 (C-Boc), 61.42 (CH₂CH₃), 55.61 (α-CH),53.56 (2 C:s) (N—CH₂), 53.48 (α-CH), 40.56 (2 C:s) (CH₂—Cl), 37.37,37.17 (CH₂-Ph), 28.34 (3 C:s) (CH₃-Boc), 14.21 (CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalanyl-L-p-amino-phenylalanine ethyl ester(20 mg; 34 μmol) was dissolved in 3 ml HCl saturated ethyl acetate. Themixture was stirred at RT for 30 minutes. The solvent was removed invacuo. The crude product was dissolved in ethanol followed byprecipitation in diethyl ether to afford JV29 (4 mg; 22%) as a lightbrown solid.

¹H NMR (CDCl₃) δ 7.46–7.29 (m, 4H, Ph-H, Phe), 7.18–7.07 (m, 2H, Ph-H,Mel), 6.78–6.66 (m, 2H, Ph-H, Mel), 4.73–4.66 (m, 1H, α-CH, Phe), 4.10(q, 2H, CH₂CH₃), 4.06–3.99 (m, 1H, α-CH, Mel), 3.79–3.56 (m, 8H,CH₂-mustard), 3.22–2.83 (m, 4H, CH₂-Ph), 1.15 (t, 3H, CH₂CH₃).

Example 10 L-Melphalanyl-D-phenylalanine ethyl ester (T4)

D-Phenylalanine (250 mg, 1.52 mmol) was dissolved in 5 ml EtOHpreviously bubbled in HCl (gas). The solution was brought to 100° C. andallowed to reflux overnight. The solvent was evaporated off affording280 mg crude product. Recrystallization from EtOH/EtOAc/pentane gave 239mg (81% yield) of D-phenylalanine ethyl ester as white crystals.

¹H NMR (CD₃OD) δ 7.4–7.22 (m, 5H, Ph-H), 4.31–4.14 (m, 3H, CH₂CH₃, α-H),3.35–3.08 (m, 2H, CH₂-Ph), 1.25–1.20 (t, 3H, CH₂CH₃).

N-tert-Butoxycarbonyl-L-melphalan (65 mg, 0.16 mmol), D-phenylalanineethyl ester (53 mg, 0.23 mmol), HOBT (32 mg, 0.24 mmol), and NMM (25 μl,0.23 mmol) were dissolved in 4 ml dichloromethane. This solution wascooled to 0° C. and EDC (44 mg, 0.23 mmol) was added. The solution wasstirred for 0.5 h at 0° C. and then for 3 h at RT. The reaction mixturewas then diluted to 20 ml with dichloromethane and the reaction stoppedby successive extractions with 10% aqueous citric acid (25 ml),saturated NaHCO₃ (25 ml) and brine (25 ml). The organic phase was thendried over anhydrous Na₂SO₄ and the solvent evaporated off under reducedpressure to give 60 mg of a yellow oil. The crude product was thenseparated by flash chromatography using a gradient eluent system ofether:pentane (2:3→1:1→1:0). The pure fractions were collected and thesolvent evaporated off to afford 25 mg (25%) ofN-tert-butoxycarbonyl-L-melphalanyl-D-phenylalanine ethyl ester as whitecrystals.

N-tert-Butoxycarbonyl-L-melphalanyl-D-phenylalanine ethyl ester (90 mg,0.16 mmol) was dissolved in 5 ml EtOAc previously bubbled with HCl(gas). The mixture was stirred at RT for 30 minutes. The solvent wasremoved in vacuo. The residue was recrystallized from EtOH/Et₂O.L-Melphalanyl-D-phenylalanine ethyl ester hydrochloride (T4) wasisolated as white crystals in 62% yield (46 mg).

Comparative Example N-Acetyl-L-melphalanyl-L-p-fluorophenylalanine ethylester (AcJ1)

L-Melphalanyl-L-p-fluorophenylalanine ethyl ester hydrochloride (10 mg,0.019 mmol), was dissolved in dichloromethane (0.5 ml) and pyridine (4μl, 0.044 mmol) was added. Acetic anhydride (2. μL, 0.22 mmol) was addedand the mixture was stirred for 1 h at RT. More pyridine (2 μl, 0.022mmol) and acetic anhydride (2. μl, 0.22 mmol) were added and a clearsolution was obtained after stirring for one hour. The solution wasextracted with 10% aqueous citric acid and brine. The organic phase wasdried over anhydrous Na₂SO₄ and the solvent evaporated off under reducedpressure. The solid residue was recrystallized in EtOH/diethyl ether toafford AcJ1 as white crystals.

¹H NMR: (CDCl₃) δ 7.09–6.90 (m, 6H, Ph-H, Phe, Mel), 6.60 (d, 2H, Ph-H,Mel), 6.25 (br d, 1H, NH), 6.04 (br d, 1H, NH), 4.73 (dd, 1H, α-CH),4.55 (dd, 1H, α-CH), 4.14 (q, 2H; CH₂CH₃), 3.76–3.58 (m, 8H,CH₂-mustard), 3.12–2.88 (m, 4H, CH₂-Ph), 1.98 (s, 3H, CH₃CO), 1.22 (t,3H, CH₂CH₃).

Biological Tests

In the following tests the cytostatic activity of the peptides of theinvention was analysed and compared to the activity of melphalan (as thehydrochloride, Alkeran injection substance, Glaxo Wellcome), P2(L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl esterhydrochloride; from Istituto Sieroterapico Milanese, Milan, Italy),sarcolysine (Istituto Sieroterapico Milanese, Milan, Italy) and a numberof standard drugs in different primary cultures of human tumor cellsfrom patients (PHTC) and human tumor cell lines. The standard drugstested were AraC (Cytosar), 5-FU (Flurablastin) and doxorubicin(Adramycin) from Pharmacia & Upjohn, vincristine (Oncovin) andvinorelbine (Navelbine) from Pierre Fabre, docetaxel (Taxotere) fromRhone Poulenc Rorer, cisplatin (Platinol) and etoposide (Vepesid) fromBristol-Myers Squibb, and topotecan from SmithKline Beecham.

The fluorometric microculture cytotoxicity assay (FMCA) (Larsson, R., etal-1992: Int J Cancer, 50, 177–185) was used to evaluate the compounds.Briefly, 96-well microtiter plates (NUNC, Roskilde, Denmark) areprepared with 20 μl drug solution at ten times the desired concentrationand stored for up to two months at −70° C. In general the substances arefirst dissolved in absolute or acidic ethanol to concentrations of 4.0to 8.2 mM and further diluted with sterile water or sterile phosphatebuffered saline (PBS, Sigma chemicals). All dilutions with water aremade directly before the experiments to minimise the influence ofmustard hydrolysis. Final ethanol concentrations do not exceed 1% v/v.At day zero of the experiment 180 μl of cell suspension of adequateconcentration is added to the wells of the thawed plate, six wells serveas controls (cell suspension only) and six wells as blanks (cell mediumonly). After 72 hours incubation the cells are washed once with PBS, and100 μl of fluorescein diacetate (10 μg/ml) in a physiological buffer isadded. After another 45 min the generated fluorescence (ex 485 nm; em528 nm) is measured in a 96-well scanning fluorometer (Fluoroscan II,Labsystems Oy, Helsinki, Finland). The generated fluorescence isproportional to the number of living cells, and data are presented assurvival index (fluorescence in test well in percent of control wellswith blank values subtracted) and IC50 (inhibitory concentration 50%, ascalculated by the software GraphPad Prism® (Graphpad Software Inc., SanDiego, Calif., USA)). Quality criteria for a successful assay include acoefficient of variation less than 30% in blank (six wells), control(six wells) and test wells (three) respectively, a control signal morethan ten times the blank and finally an initial cell viability of morethan 70% (primary human tumour cultures) or 90% (cell lines) as judgedby the trypan blue exclusion test.

Fluorescein diacetate (FDA, Sigma) was dissolved in DMSO to 10 mg/ml andkept frozen as a stock solution in the dark. Cell growth mediumRPMI-1640 (Sigma) supplemented with 10% heat-inactivated fetal calfserum (FCS, Sigma chemical Co., St. Louis, Mo.), 2 mM glutamine, 100μg/ml streptomycin, and 100 μg/ml penicillin, was used.

Much work with FMCA has focused on prediction of clinical activity ofanticancer drugs for individual patients. The predictive ability of thisassay for this application has been shown to be comparable to that ofmany other clinically accepted test procedures with sensitivity andspecificity in the range of 80–90, and 60–70, respectively.

Test 1. Cytotoxic Activity in Fresh Human Tumour Specimens

The cytotoxic activity of melphalan, P2(L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl esterhydrochloride) and J1 (L-melphalanyl-p-L-fluorophenylalanine ethyl esterhydrochloride) on human tumour samples was compared using thefluorometric microculture cytotoxicity assay (FMCA). Each drug wastested in six concentrations ranging from 82 to 0.131 μM, eachconcentration in triplicate.

In total twenty-seven fresh human tumour specimens were analysed, thesewere of different origin: fourteen hematological (of which at least fivewere previously treated with cytotoxic drugs in the clinic) and thirteensolid tumour (five previously treated) samples were analysed.Concentration-response curves were plotted and the IC50s weredetermined. The results are presented in FIG. 2, wherein the diagonalsolid lines represent equipotency, and dots above this line favour thedrug on the x-axis and vice versa. The result shows that J1 in all caseswas more active than P2, which in turn was more active than melphalan.

Using the same FMCA assay the cytotoxic activity of melphalan, P2 and J1was again tested, this time on in total sixty-four fresh human tumourspecimens. The tumours were of different origin: forty-two hematologicaland twenty-two solid tumour samples were analysed. In parallel theactivity of some clinically well known and used standard substances wasassayed at concentrations of from 0.92 to 10.3 μM. The standard drugswere araC, vincristin, vinorelbine, docetaxel, cisplatin, doxorubicin,and etoposide. A comparison of the activities is presented as survivalindex at a defined concentration in Table 1 below. J1 shows a superioractivity at 0.66 μM.

TABLE 1 Activity of J1, melphalan, P2 and standard drugs in PHTC Tumorcell survival index % Concentration Hematological Solid Drug μM (n = 42)(n = 22) J1 0.66 13.7 46.9 Melphalan 3.3 41 88.8 P2 0.66 34.7 86.0 AraC10.3 — 102 Vincristine 3.0 39 93 Vinorelbine 3.1 50 73 Docetaxel 6.2 —67 Cisplatin 6.7 63 79 Doxorubicin 0.92 29 97 Etoposide 8.5 47 81Test 2. Cytotoxic Activity in a Panel of Ten Human Tumour Cell Lines

The cytotoxic activity of nine different peptides of the invention(prepared in Examples 1–9) was compared with melphalan, P2(L-prolyl-m-L-sarcolysyl-p-L-fluorophenylalanine ethyl esterhydrochloride), sarcolysine, and the standard drugs doxorubicin,vincristine, cisplatin, 5-Fu, and topotecan in ten cell lines using theFMCA method. Each drug was tested in six concentrations ranging from 40to 0.013 μM (melphalan from 1.6 mM to 0.51 μM), and each concentrationin duplicate. The experiment was repeated three times and all survivaldata were then used to calculate IC50 for each drug on each cell line.

The selection of cells in the cell line panel has been described earlier(Dhar, S., et al. 1996: Br J Cancer, 74, 888–896). Four parental linesof different origin (lymphoma U-937 GBT; myeloma RPMI 8226; small celllung cancer NCI-H69; and leukemia CCRF-CEM), five sublines selected forvarious drugs, and one primary resistant cell line (renal carcinomaACHN) was included. The subline U-937-vcr is selected for vincristineresistance, subline 8226Dox40 and H69AR for doxorubicin resistance,subline 8226LR5 for melphalan resistance. The cell line growth andmorphology were monitored on a weekly basis, resistance every two orthree months.

The results are presented in Table 2 and show that the cytotoxicactivity varies among the peptides and compare favourably withmelphalan, m-L-sarcolysine and P2. In addition some of the peptides showhigher activities on certain cell lines than some of the tested standardchemotherapeutic substances.

TABLE 2 Activity of peptides of the invention compared to melphalan,sarcolysine, P2 and standard drugs in a cell line panel representingdefined forms of cytotoxic drug resistance, IC50 (μM) Cell line J1 J3JV22 JV24 JV25 JV26 JV27 JV28 JV29 CEM/S 0.03 0.06 0.02 0.05 0.04 0.050.14 0.06 0.06 CEM/R 0.01 0.02 0.01 0.03 0.02 0.03 0.1 0.04 0.03 ACHN0.4 1.72 0.17 0.32 0.2 0.22 0.21 0.05 0.19 H69 0.04 0.2 0.04 0.08 0.040.06 0.29 0.05 0.09 H69AR 0.9 1.36 0.39 0.48 0.28 0.51 1.4 0.37 0.468226S 0.81 1.51 0.67 1.11 0.65 1.05 1.01 0.51 0.9 8226Dox40 1.76 5.661.38 5.54 1.2 1.73 1.58 0.89 2.01 8226LR5 2.47 3.52 1.86 2.5 1.82 1.992.46 1.88 2.73 U937gtb 0.07 0.23 0.14 0.2 0.15 0.23 0.16 0.1 0.15U937vcr 0.11 0.42 0.13 0.21 0.16 0.26 0.26 0.15 0.19 MEAN 0.66 1.47 0.481.05 0.46 0.61 0.76 0.41 0.68 Cell line Melphalan P2 SarcolysineDoxorubicin Vincristine Cisplatin 5-FU Topotecan CEM/S 1.48 0.94 2.540.18 0.08 2.50 57.4 0.02 CEM/R 0.94 0.63 1.70 1.31 0.01 1.87 44.5 0.02ACHN 133.32 4.59 390 14.2 109 17.8 769 16.3 H69 13.44 4.59 41.15 1.6669.8 92.47 769 1.72 H69AR 48.74 4.27 61.4 11.6 121 13.5 769 180.5 8226S10.82 5.11 30.4 0.13 0.01 14.8 75.6 0.87 8226Dox40 35.18 5.43 41.38 4.970.9 15.66 60.6 0.59 8226LR5 30.61 7.4 31.88 0.55 0.08 16.33 5.30 0.21U937gtb 1.8 1.78 3.78 0.11 0.01 2.57 88.4 0.02 U937vcr 2.95 1.77 6.640.42 0.08 3.0 572.0 0.02 MEAN 27.93 3.35 61.09 3.51 30.1 18.0 321 20.0Test 3. Cytotoxic Activity of J1 in Lung Cancer Cell Lines

J1 was also tested against a panel of lung cancer cell lines andcompared with melphalan and some standard chemotherapeutic agents usingthe FMCA. In total four small cell lung cancer cell lines (U-1906 L andE, U-1285, and U-1690) and seven non-small cell lung cancer cell lines(NCI-H23, U-1752, NCI-H611, NCI-H157, U-1810, NCI-H125 and U-1568) wereanalyzed. The results are presented in Table 3.

TABLE 3 Cytotoxic activity in lung cell cancer lines, IC50 (μM) Cis-Doxo- Cell line J1 Melphalan platin rubicin Docetaxel Topotecan NCI-H237.6 125 11.6 0.14 1.5 5.84 U-1752 1.3 39.3 11.7 0.42 0.46 28.0 NCI-H6113.17 78.3 50.7 183 1.1 236 NCI-H157 3.9 125 27.0 0.2 0.1 4.33 U-18100.01 1.0 10.3 22.8 0.1 0.08 NCI-H125 9.73 85.2 7.0 0.5 0.16 236 U-15681.64 34.7 23.4 183 5.94 115 U1906L 0.22 36.7 12.2 0.92 0.1 0.12 U12850.04 1.97 2.93 0.52 0.1 0.17 U-1906E 1.17 20.9 21.1 0.44 0.1 0.08 U16901.39 12.4 6.73 2.21 7.7 0.92The results show that J1 on a molar basis compares favourably withmelphalan and most standard agents used in the clinical therapy of lungcancer and also shows an overall activity similar to that observed fordocetaxel, a tubulin active drug which lately has been introduced intolung cancer therapy. Since J1 and other melphalan derivatives arenon-cross resistant with taxanes (judged from correlation analysis ofthe activity patterns) these two drug classes may form attractivetreatment combinations.Test 4. Cytotoxic Activity of J3 and JV28 in Primary Cultures of HumanTumor Cells from Patients (PHTC)

In another set of experiments the activity of J1, J3 and JV28 wascompared with that of melphalan, sarcolysine and P2 in 31 PHTC culturesusing the FMCA. The results are presented in Table 4.

TABLE 4 Cytotoxic activity of drugs in PHTC Tumor cell survival index(%) Hematological Solid tumors tumors Total Drug Conc. μM n = 7–11 n =12–20 n = 19–31 J3 4 7 36.3 25.9 JV28 4 6.7 44.2 30.4 J1 4 6.6 56.5 40.9Melphalan 10 47.6 91.0 73.4 Sarcolysine 10 47.1 93.2 76.2 P2 4 7 68.946.1The results show that J3 and JV28 were significantly (P<0.001 Studentst-test) more active against hematological PHTC samples than bothmelphalan and sarcolysine. J3 and JV28 were also significantly (P<0.01)more active against solid tumor samples compared not only to melphalanand sarcolysine but also compared to P2. The difference between J1 andmelphalan as well as sarcolysine and P2 was statistically significantfor both hematological and solid tumours. The results demonstrates anunexpectedly high activity of the new peptides against solid tumours asa group which was not shared by the reference compounds.Test 5. Cytotoxic Activity in Different Tumour Models

The tumour type specific spectrum of antitumour activity was analysed incell lines and primary cultures of human tumour cells from patients.Although melphalan has been classified as active against allhematological diagnoses no activity, i.e. <50% reduction in tumour cellsurvival, was observed against the solid tumour types. J3. J1 and JV28,on the other hand, showed activity against several types of solidtumours including breast cancer, ovarian carcinoma and lung cancer. Thedifference in tumor type specific activity between J1, J3 and JV 28versus P2 is depicted in Table 5.

TABLE 5 Cytotoxic activity in different tumor models Median response at4.0 μM Tumor cell >50% reduction in tumor cell survival system Number P2J1 J3 JV28 Ovarian 8 No Yes Yes Yes carcinoma Breast 4 No Yes Yes Yescancer NSC Lung 8 No Yes Yes Yes cancer Prostate 1 No not done Yes Yescancer Corpus 4 No No Yes not done cancer Adenocarcinoma 5 No Yes YesYesWhereas P2 was determined inactive in the listed diagnoses of Table 5,J3 was found active. J1 and JV28 were found to be active in most ofthese diagnoses. These data show that the tumour type specific spectrumof activity for J1, J3 and JV28 is broader than for the referencecompounds and includes important types of solid tumours.Test 6. Circumvention of Melphalan Resistance

The ability to circumvent melphalan resistance was investigated in 27PHTC samples from 9 patients with hematological and 18 patients withsolid tumours. The samples were classified according to melphalanresistance based on FMCA test results (Survival Index) from severalhundred PHTC samples previously tested for melphalan. These data wereused to establish cut-off lines to divide samples into three categories:Low, Intermediate or Extreme drug resistance (LDR, IDR and EDR,respectively) using the median value and median+1 standard deviationfrom that database. Melphalan and the new compounds J1, J3, and JV28were subsequently classified into these groups. The classification ofthe melphalan resistance was performed according to the principlesdescribed by Larsson and Nygren, Anticancer Research 13; 1825–1830,1993. The new peptides and P2 were tested at a concentration of 4 μM andmelphalan at 10 μM. The results of the study is presented in Table 6below.

TABLE 6 Circumvention of melphalan resistance Number of samples (%) LDRIDR EDR Total Melphalan  8 (30%) 10 (37%) 9 (33%) 27 J1 13 (76%)  3(18%) 1 (6%) 17 J3 23 (85%)  4 (15%) 0 (0%) 27 JV28 14 (82%)  2 (12%) 1(6%) 17 P2 14 (74%)  4 (21%) 1 (5%) 19The results show that significantly fewer samples of the new peptideswere classified as IDR and EDR indicating that these drugs have theability to circumvent melphalan resistance. In this analysis it isassumed that at least half of the melphalan concentration may be reachedin the clinical setting with the new peptides.Test 7. Comparing the Activity of J1 and AcJ1

In order to investigate the difference in activity between a compoundhaving a free amino group at the N-terminus and the same compound havingsaid amino group protected, the acetylated form of J1 was synthesisedand the activity compared with J1 using the FMCA procedure describedabove on the U-937 and RPMI 8226S cell lines. The results showed aseveral-fold lower potency (higher IC50; 0.61 and 2.9 μM respectively)for the acetylated form compared to J1 having a free amine, indicating apreference for the latter form with respect to antitumour activity.

Conclusions

The overall results demonstrate a significantly higher activity of thenew compounds constituting the invention compared to all referencecompounds (melphalan, sarcolysin and P2). Furthermore the novelcompounds also demonstrated a broader spectrum of tumour type specificactivity compared to the reference compounds with documented activityobserved for many solid tumour diagnoses. Since these compounds alsoshow a similar or slightly better relative activity in malignant vsnormal cells, judged by taking the ratio of IC50s obtained in malignant(chronic lymphocytic leukemias) over normal lymphocytes, the clinicalpotential of these new compounds must be considered to be high.

1. A di- or tripepetide having the formula I:

wherein R₁ is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂,alkylamino, cycloalkylamino, or arylamino; R₂ is

wherein R₃ is independently NH₂, OH, O-alkyl, N-alkyl, O-acyl, NH-acyl,N(CH₂CH₂Cl )₂, NO₂, F, CF₃ or H, and n is 1 or 2; X is

wherein R₅ is H; R₄ is a natural or modified cyclic or aromatic aminoacid, or H; and pharmaceutically acceptable salts thereof.
 2. A di- ortripepetide having the formula V:

wherein R₁ is alkyloxy, cycloalkyloxy, aryloxy, arylalkyloxy, NH₂,alkylamino, cycloalkylamino, or arylamino; R₃ is NH₂, OH, O-alkyl,N-alkyl, O-acyl, NH-acyl, N(CH₂CH₂Cl)₂, NO₂, F, CF₃ or H: and R₄ is anatural or modified cyclic or aromatic amino acid, or H; andpharmaceutically acceptable salts thereof.
 3. A method of manufacturinga medicament, comprising: formulating the peptide according to claim 1with a pharmaceutically acceptable excipient.
 4. The peptide accordingto claim 1, wherein R₃ is F.
 5. The peptide according to claim 2,wherein R₁ is alkyloxy; R₃ is F, CF₃, H, OH, O-alkyl, NO₂, N(CH₂CH₂Cl)₂,NH-acyl or NH₂; and R₄ is H.
 6. The peptide according to claim 5, whichis L-melphanyl-p-L-fluoro-phenylalanine ethyl ester or apharmaceutically acceptable salt thereof.
 7. The peptide according toclaim 5, which is L-melphanyl-p-L-fluoro-phenylalanine isopropyl esteror a pharmaceutically acceptable salt thereof.
 8. The peptide accordingto claim 2, wherein R₁ is alkyloxy; R₃ is F, CF₃, H, OH, O-alkyl,NH-acyl, NO₂, N(CH₂CH₂Cl)₂ or NH₂; and R₄ is a natural or modifiedcyclic or aromatic amino acid.
 9. The peptide according to claim 8,which is L-prolin-L-melphanyl-p-fluorophenylalanine ethyl ester or apharmaceutically acceptable salt thereof.
 10. A pharmaceuticalcomposition for the treatment of malignant tumours, comprising at leastone peptide compound according to claim 1, together with at least onepharmaceutically acceptable carrier and/or excipient.
 11. Thepharmaceutical composition according to claim 10, for the treatment ofbreast cancer, lung cancer, ovarian cancer, leukemias, lymphomas andmultiple myeloma.
 12. A method of treating malignant tumours,comprising: administering a pharmaceutically effective dose of thepeptide according to claim 1 to a subject in need thereof.
 13. A methodof manufacturing a medicament, comprising: formulating the peptideaccording to claim 2 with a pharmaceutically acceptable excipient. 14.The peptide according to claim 2, wherein R₃ is F.
 15. A pharmaceuticalcomposition for the treatment of malignant tumors, comprising at leastpeptide compound according to claim 2, together with at least onepharmaceutically acceptable carrier and/or excipient.
 16. Thepharmaceutical composition according to claim 15, for the treatment ofbreast cancer, lung cancer, ovarian cancer, leukemias, lymphomas andmultiple myeloma.
 17. A method of treating malignant tumours,comprising: administering a pharmaceutically effective dose of thepeptide according to claim 2 to a subject in need thereof.